The macrolide antibiotic erythromycin interacts with bacterial 23S ribosomal RNA (rRNA) making contacts that are limited to hairpin 35 in domain II of the rRNA and to the peptidyl transferase loop in domain V. These two regions are probably folded close together in the 23S rRNA tertiary structure and form a binding pocket for macrolides and other drug types. Erythromycin has been derivatized by replacing the L-cladinose moiety at position 3 by a keto group (forming the ketolide antibiotics) and by an alkyl-aryl extension at positions 11/12 of the lactone ring. All the drugs footprint identically within the peptidyl transferase loop, giving protection against chemical modification at A2058, A2059 and G2505, and enhancing the accessibility of A2062. However, the ketolide derivatives bind to ribosomes with widely varying affinities compared with erythromycin. This variation correlates with differences in the hairpin 35 footprints. Erythromycin enhances the modification at position A752. Removal of cladinose lowers drug binding 70-fold, with concomitant loss of the A752 footprint. However, the 11/12 extension strengthens binding 10-fold, and position A752 becomes protected. These findings indicate how drug derivatization can improve the inhibition of bacteria that have macrolide resistance conferred by changes in the peptidyl transferase loop.